US20190367954A1 - Method for producing branched aldehydes - Google Patents
Method for producing branched aldehydes Download PDFInfo
- Publication number
- US20190367954A1 US20190367954A1 US15/779,214 US201515779214A US2019367954A1 US 20190367954 A1 US20190367954 A1 US 20190367954A1 US 201515779214 A US201515779214 A US 201515779214A US 2019367954 A1 US2019367954 A1 US 2019367954A1
- Authority
- US
- United States
- Prior art keywords
- carboxylic acids
- free
- reduction
- aldehydes
- bound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000001299 aldehydes Chemical class 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000203 mixture Substances 0.000 claims abstract description 27
- 150000001735 carboxylic acids Chemical class 0.000 claims abstract description 23
- 150000001298 alcohols Chemical class 0.000 claims abstract description 19
- 239000000126 substance Substances 0.000 claims abstract description 17
- 239000002028 Biomass Substances 0.000 claims abstract description 12
- 241001480517 Conidiobolus Species 0.000 claims abstract description 11
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 11
- 241000233866 Fungi Species 0.000 claims abstract description 8
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 8
- 239000007800 oxidant agent Substances 0.000 claims abstract description 8
- 230000002452 interceptive effect Effects 0.000 claims abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 3
- 239000006227 byproduct Substances 0.000 claims abstract 2
- 239000000284 extract Substances 0.000 claims description 30
- 230000009467 reduction Effects 0.000 claims description 30
- 102000004190 Enzymes Human genes 0.000 claims description 16
- 108090000790 Enzymes Proteins 0.000 claims description 16
- OQWNKUAZQSLNSR-UHFFFAOYSA-N 12-methyltridecanal Chemical compound CC(C)CCCCCCCCCCC=O OQWNKUAZQSLNSR-UHFFFAOYSA-N 0.000 claims description 15
- GDOPTJXRTPNYNR-UHFFFAOYSA-N methyl-cyclopentane Natural products CC1CCCC1 GDOPTJXRTPNYNR-UHFFFAOYSA-N 0.000 claims description 12
- 230000003647 oxidation Effects 0.000 claims description 12
- 238000007254 oxidation reaction Methods 0.000 claims description 12
- 230000007062 hydrolysis Effects 0.000 claims description 11
- 238000006460 hydrolysis reaction Methods 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 11
- 238000004821 distillation Methods 0.000 claims description 10
- 108050006958 Alpha-dioxygenases Proteins 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 8
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 claims description 8
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 claims description 7
- 241000142142 Conidiobolus heterosporus Species 0.000 claims description 7
- 239000012141 concentrate Substances 0.000 claims description 7
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 claims description 4
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 claims description 4
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 4
- 229910052744 lithium Inorganic materials 0.000 claims description 4
- 241000141888 Conidiobolus denaeosporus Species 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- LOSARDBKYQAFPN-UHFFFAOYSA-N 12-methyltetradecanal Chemical compound CCC(C)CCCCCCCCCCC=O LOSARDBKYQAFPN-UHFFFAOYSA-N 0.000 claims description 2
- IKBYSGSLCRPWRO-UHFFFAOYSA-N 14-methylpentadecanal Chemical compound CC(C)CCCCCCCCCCCCC=O IKBYSGSLCRPWRO-UHFFFAOYSA-N 0.000 claims description 2
- PCMJMKCAWAECPE-UHFFFAOYSA-N 16-methyloctadecanal Chemical compound CCC(C)CCCCCCCCCCCCCCC=O PCMJMKCAWAECPE-UHFFFAOYSA-N 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 239000012279 sodium borohydride Substances 0.000 claims description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 2
- 238000000605 extraction Methods 0.000 description 30
- 238000006243 chemical reaction Methods 0.000 description 26
- 238000006722 reduction reaction Methods 0.000 description 21
- 235000014113 dietary fatty acids Nutrition 0.000 description 18
- 229930195729 fatty acid Natural products 0.000 description 18
- 239000000194 fatty acid Substances 0.000 description 18
- 235000019197 fats Nutrition 0.000 description 17
- 150000004665 fatty acids Chemical class 0.000 description 16
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 16
- IPKIIZQGCWXJFM-UHFFFAOYSA-N 2-methyl-1-(4-nitrophenyl)sulfonylaziridine Chemical compound CC1CN1S(=O)(=O)C1=CC=C([N+]([O-])=O)C=C1 IPKIIZQGCWXJFM-UHFFFAOYSA-N 0.000 description 15
- YYVJAABUJYRQJO-UHFFFAOYSA-N Isomyristic acid Natural products CC(C)CCCCCCCCCCC(O)=O YYVJAABUJYRQJO-UHFFFAOYSA-N 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 102000004882 Lipase Human genes 0.000 description 12
- 108090001060 Lipase Proteins 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 10
- 239000004367 Lipase Substances 0.000 description 10
- 239000008272 agar Substances 0.000 description 10
- 235000019421 lipase Nutrition 0.000 description 10
- 150000002632 lipids Chemical class 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 229960000318 kanamycin Drugs 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- -1 acetone) Chemical class 0.000 description 5
- 230000036983 biotransformation Effects 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 230000002538 fungal effect Effects 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 125000005456 glyceride group Chemical group 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 241001197104 Nocardia iowensis Species 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 4
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 241001480501 Entomophthoraceae Species 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000892564 Ulmus parvifolia Species 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- XKLJLHAPJBUBNL-UHFFFAOYSA-N 12-methyltetradecanoic acid Chemical compound CCC(C)CCCCCCCCCCC(O)=O XKLJLHAPJBUBNL-UHFFFAOYSA-N 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229910010084 LiAlH4 Inorganic materials 0.000 description 2
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000179532 [Candida] cylindracea Species 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- ZQPPMHVWECSIRJ-MDZDMXLPSA-N elaidic acid Chemical compound CCCCCCCC\C=C\CCCCCCCC(O)=O ZQPPMHVWECSIRJ-MDZDMXLPSA-N 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 239000012280 lithium aluminium hydride Substances 0.000 description 2
- 238000002803 maceration Methods 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000005325 percolation Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- MGSRCZKZVOBKFT-UHFFFAOYSA-N thymol Chemical compound CC(C)C1=CC=C(C)C=C1O MGSRCZKZVOBKFT-UHFFFAOYSA-N 0.000 description 2
- SZHOJFHSIKHZHA-UHFFFAOYSA-N tridecanoic acid Chemical compound CCCCCCCCCCCCC(O)=O SZHOJFHSIKHZHA-UHFFFAOYSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- ZONJATNKKGGVSU-UHFFFAOYSA-N 14-methylpentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCC(O)=O ZONJATNKKGGVSU-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- YZKLUEWQADEGKP-UHFFFAOYSA-N 5-(2,4-dichlorophenyl)cyclohexane-1,3-dione Chemical compound ClC1=CC(Cl)=CC=C1C1CC(=O)CC(=O)C1 YZKLUEWQADEGKP-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241001480514 Ancylistaceae Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- QYTDEUPAUMOIOP-UHFFFAOYSA-N CC1(C)CCCC(C)(C)N1[O] Chemical compound CC1(C)CCCC(C)(C)N1[O] QYTDEUPAUMOIOP-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000222175 Diutina rugosa Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000235577 Entomophthorales Species 0.000 description 1
- 241001510830 Entomophthoromycotina Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241001588792 Heteropsorus Species 0.000 description 1
- YITMLDIGEJSENC-UHFFFAOYSA-N Hexadecen Natural products CCCCCCCCCCCCCC=CC YITMLDIGEJSENC-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- ZOCYQVNGROEVLU-UHFFFAOYSA-N Isopentadecylic acid Natural products CC(C)CCCCCCCCCCCC(O)=O ZOCYQVNGROEVLU-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710084376 Lipase 3 Proteins 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000187492 Mycobacterium marinum Species 0.000 description 1
- 241000187488 Mycobacterium sp. Species 0.000 description 1
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 229910019093 NaOCl Inorganic materials 0.000 description 1
- 241000187681 Nocardia sp. Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical class CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 1
- 108091007187 Reductases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 239000005844 Thymol Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 235000001547 Ulmus pumila Nutrition 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000004362 fungal culture Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- YAQXGBBDJYBXKL-UHFFFAOYSA-N iron(2+);1,10-phenanthroline;dicyanide Chemical compound [Fe+2].N#[C-].N#[C-].C1=CN=C2C3=NC=CC=C3C=CC2=C1.C1=CN=C2C3=NC=CC=C3C=CC2=C1 YAQXGBBDJYBXKL-UHFFFAOYSA-N 0.000 description 1
- IIUXHTGBZYEGHI-UHFFFAOYSA-N iso-margaric acid Natural products CC(C)CCCCCCCCCCCCCC(O)=O IIUXHTGBZYEGHI-UHFFFAOYSA-N 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000000956 solid--liquid extraction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960000790 thymol Drugs 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/205—Heterocyclic compounds
- A23L27/2052—Heterocyclic compounds having oxygen or sulfur as the only hetero atoms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/26—Meat flavours
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/27—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
Definitions
- the invention is in the field of flavoring substances and relates to a new method for the production of branched aldehydes, in particular 12-methyltridecanal.
- Branched aldehydes are interesting odorous and flavoring substances that are prevalent in nature. 12-methyltridecanal in particular is an important building block for authentically tasting bovine flavors and can currently only be commercially produced by chemical synthesis (cf. Kerscher, et. al., J. Agric. Food Chem. 48(6), p. 2387-2390, 2000).
- the objective of the present invention was therefore to provide a method for the production of branched aldehydes in general and 12-methyltridecanal in particular by biotechnological and therefore natural means.
- a first object of the invention relates to a method for the production of branched aldehydes, preferably those comprising 12 to 18 carbon atoms and/or having a methyl branching, in particular 12-methyltridecanal, 12-methyltetradecanal, 14-methylpentadecanal, 16-methyloctadecanal or mixtures thereof, the method comprising the following steps:
- the organisms which, due to the fatty acid spectrum they produce, serve as suitable bioreactors for the production of branched fatty acids of the desired chain length belong to a special genus of fungi, namely Conidiobolus , which mainly comprises saprobions that degrade dead plant material or live in the soil.
- Conidiobolus which mainly comprises saprobions that degrade dead plant material or live in the soil.
- the two preferred species C. denaeosporus and C. heterosporus are described in connection with decomposition of leaves of the Chinese elm ( Ulmus parvifolia ). Taxonomically, the two genera are classified as follows:
- Fungal mycelium/mycelia can be cultivated in a known manner using a suitable nutrient medium in solid or liquid culture. Agar plates produced with a nutrient solution of malt and yeast extract have proved their worth.
- biomass After the cultures have formed a sufficient amount of biomass, the biomass, if necessary, is separated from the nutrient solution for example by centrifuging, filtration or other suitable separation methods. This is followed by extraction of carboxylic acids in their free or bound form. Solid cultures, such as those obtained after freeze-drying, can be extracted directly.
- extracts may be produced in a manner known per se, i.e. for example by aqueous, alcoholic or aqueous-alcoholic extraction of the cultures themselves or dry mass obtained therefrom. All conventional extraction methods are suitable, e.g. maceration, remaceration, digestion, motion maceration, vortex extraction, ultrasonic extraction, countercurrent extraction, percolation, repercolation, evacolation (extraction under reduced pressure), diacolation or solid-liquid extraction under continuous backflow.
- the percolation method is advantageous for large-scale use.
- Organic solvents, water (preferably hot water at a temperature above 80° C. and in particular above 95° C.) or mixtures of organic solvents and water, in particular low-molecular weight alcohols with more or less high water contents, may be used as solvents for carrying out the extractions.
- Extraction with methanol, ethanol, pentane, hexane, heptane, acetone, propylene glycols, polyethylene glycols, ethyl acetate and mixtures thereof and their aqueous mixtures is particularly preferred.
- the extraction usually takes place at 20 to 100° C., preferably at 30 to 90° C., in particular at 60 to 80° C.
- extraction is carried out in an inert gas atmosphere to avoid oxidation of the active ingredients of the extract. This is particularly important for extractions at temperatures above 40° C.
- the durations of extraction are set by the skilled person depending on the starting material, the extraction method, the extraction temperature, the ratio of solvent to raw material and others.
- the raw extracts obtained may undergo further conventional steps, such as purification, concentration and/or discoloration.
- the extracts formed in this way can, for example, be subjected to a selective separation of individual undesirable ingredients.
- the extraction can occur to any degree of extraction, but is usually carried out until exhaustion.
- the present invention encompasses the finding that the extraction conditions and the yields of the final extracts can be selected by the skilled person according to any desired field of application.
- the extracts which are hereinafter also referred to as the first intermediate, may be present as aqueous preparations and/or preparations dissolved in organic solvents as well as spray-dried or freeze-dried, anhydrous solids.
- aliphatic or branched alcohols with 1 to 6 carbon atoms e.g. ethanol, 2-methoxy-2-methylpropane
- ketones e.g. acetone
- halogenated hydrocarbons e.g. chloroform or methylene chloride
- lower esters or polyols e.g. glycerol or glycols
- the extracts can be subjected to hydrolysis before the reduction to release the fraction of fatty acids bound as glyceride.
- the hydrolysis can occur chemically, but a biological alternative is preferable. This alternative can occur microbially, i.e. using living microorganisms, as well as enzymatically, since these hydrolyses are gentler and also offer regulatory advantages.
- Hydrolases from Candida in particular Candida rugosa or Candida cylindracea are suitable for the biological alternative.
- a first intermediate of a mixture of different linear and branched, saturated and unsaturated fatty acids, which can also be bound as glycerides is obtained.
- Such a mixture may include, for example, the following representatives: tridecanoic acid, 12-methyltridecanoic acid, tetradecanoic acid, 12-methyltetradecanoic acid, pentadecanoic acid, 14-methylpentadecanoic acid, hexadecanoic acid, hexadecen(9)acid, 15-methylhexadecanoic acid, heptadecanoic acid, elaidic acid, oleic acid and arachidonic acid.
- the mixture of fatty acids or fatty acid glycerides is now subjected to a reduction. Like hydrolysis, this can be done chemically, but preferably a biological alternative is chosen, as this then leads to a production method that is considered natural in the sense of European flavoring legislation.
- the preferred biological alternative can be carried out with enzymes as well as with cell lysates, cell extracts or whole cells that have the enzymatic activity required for the reduction.
- the reaction products can be extracted from the reaction mixture using extraction methods known to the skilled person. After removal of the extractant, the concentrate obtained can then be taken up in the desired concentration with the solvent of choice.
- the chemical reduction of the mixture of fatty acids respectively fatty acid glycerides can be carried out with sodium borohydride, but lithium alanate should preferably be used.
- the acid or ester functions are reduced to hydroxyl groups; the second, in this case chemical intermediate, thus completely or at least predominantly contains the corresponding alcohols of the linear and branched carboxylic acids.
- yields of 85% to 95% of the theoretical yield are achieved, which can be increased if the reduction is followed by another distillation step, for example a ball tube (bulb to bulb; Kugelrohr) distillation.
- the yield is around 25 to 35% based on the total amount of reduction products.
- the reduction also results in the saturation of unsaturated species contained in the mixture.
- Reduction methods using hydrides belong to the standard reactions of a preparative organic chemists and represent textbook knowledge that therefore requires no further comprehensive explanation.
- the reduction is preferably carried out at temperatures in the range of 15 to 25° C., whereby, as expected, a temporary temperature increase occurs at the beginning of the reaction.
- the second chemical intermediate serves as a starting material for the subsequent oxidation, during which the alcohols are converted into the corresponding aldehydes.
- TEMPO is preferably used as an oxidizing agent.
- TEMPO stands for 2,2,6,6-tetramethylpiperidinyloxyl.
- this radical is not thermodynamically stable, its comparatively high persistence is due to substituents that influence the service life through steric effects.
- the substituents are located in the vicinity of the radical electron, so that it has an average life of one minute in an oxygen-free solution.
- TEMPO TEMPO together with a suitable co-oxidant for the two-phase oxidation of alcohols is described by Anelli et al. in J Organic Chemistry. 52, p. 2559-2562 (1987) (“Anelli oxidation”) and used for example in Organic Syntheses, Coll. Vol. 8, p. 367 (1993); Vol. 69 p. 212 (1990).
- TEMPO in combination with two co-oxidants, namely an alkali bromide and an alkali hypochlorite.
- a combination of potassium bromide and sodium hypochlorite is preferred here.
- a molar ratio of TEMPO, alkali bromide and alkali hypochlorite of about 1:(2 to 10):(10 to 40), particularly about 1:(2 to 10):(10 to 40) and more particularly of about 1:(4 to 8):(15 to 30) has proven its worth. Since the reaction is highly exothermic, it is recommended to keep the reaction temperature in the range of about ⁇ 5 to +10° C.
- enzymes such as commercially available aldehyde dehydrogenases (ALDH, EC 1.2.1.x) and carboxylic acid reductases, e.g. from Norcardia sp. (Aimin He, Tao Li, Lacy Daniels, Ian Fotheringham, John P. N. Rosazza Appl Environ Microbiol. 2004 March; 70(3): 18744881) and/or Mycobacterium marinum (M. Kalim Akhtar, Nicholas J. Turner, Patrik R. Jones Proc Natl Acad Sci USA. 2013 Jan. 2; 110(1): 87-92), can be used.
- Carboxylic acid reductases can be obtained by homologous expression in Nocardia sp.
- Nocardia iowensis in particular Nocardia iowensis , as well as heterologously by recombinant expression in suitable host organisms in particular E. coli , but preferably also by cultivation of Nocardia iowensis , which as so-called wild-type strain also expresses the enzymes.
- suitable host organisms in particular E. coli
- Nocardia iowensis which as so-called wild-type strain also expresses the enzymes.
- the enzymes are present in purified form, are only partially concentrated, or are present in a cell raw extract or in native or recombinant cells.
- a further object of the invention concerns a concentrate of methyl-branched aldehydes having 12 to 18 carbon atoms, obtainable by the method described above, which concentrate can contain the methyl-branched aldehydes in amounts of 10 to 100, preferably 25 to 95 and in particular about 40 to about 60% by weight.
- the concentrates can be obtained directly on the basis of the chemically formed second intermediate (f), the biologically formed second intermediate (e) or the completely or partially purified products (g) by suitable, preferably gentle drying methods, such as spray drying or freeze drying.
- suitable, preferably gentle drying methods such as spray drying or freeze drying.
- the difference to 100% by weight may be due to carriers and/or other flavoring substances.
- concentrated aqueous solutions can also be sold.
- a final object of the present invention concerns the use of the concentrates or alternatively the products directly obtained by the process as described above as flavoring components, in particular as flavoring components for foodstuffs, in order to give them a beef flavor.
- Table 1 below gives an overview of the fungi examined, their exact designation, number of the strain collection (CBS number), the isolation substrate and the isolation site.
- the fungal strains were cultivated on malt extract agar plates. To produce the plates, the media components were weighed (Table 2), filled up with distilled water to 1 litre and autoclaved (120° C., 20 min). A 1 cm 2 mycelium-covered piece of agar from the vital peripheral area of a plated culture was placed in the middle of the agar plates, the petri dishes were sealed with parafilm and then incubated with or preferably without light at 24° C. in the incubator. After about 5 days (CHET-N) and 14 days (CHET-U and CDE), pre-cultures were inoculated from the plates that were about three-quarters overgrown. In parallel, further agar plates for the next pre-cultures were inoculated according to the same principle. Table 2 shows the composition of the malt extract-yeast extract-agar for the cultivation of Conidiobolus strains:
- a 1 cm 2 agar piece of the outer mycelium was transferred under sterile conditions to a 250 mL Erlenmeyer flask filled with 100 mL culture medium and treated with an ultraturrax dispersing rod (30 s, 9,800 rpm).
- the pre-cultures were incubated at 24° C. in an incubator (Multitron, Infors, Einsbach; 24° C., 150 rpm, deflection 25 mm) with no light.
- Main culture For cultivation of the main cultures, the pre-culture was treated under sterile conditions with an ultraturrax dispersing rod (30 s, 9,800 rpm). 20 mL of this suspension was added to 200 mL fresh culture medium in a 500 mL Erlenmeyer flask. Cultivation took place at 24° C. with exclusion of light in an incubation shaker (24° C., 150 rpm, deflection 25 mm).
- the main culture was filtered through a filter paper (range of retention 8-12 ⁇ m) at the end of the cultivation period.
- the residue was mixed with about 200 mL 4 M HCl, heated to boiling point and digested for 30 min in the boiling heat.
- the solution was then filtered through a pleated filter while still hot and washed with hot water until the wash water was neutral.
- the filter paper was extracted for 4 h with petroleum gasoline (boiling range 40-60° C.) using a Soxhlet apparatus. After removing the solvent with a rotary evaporator, the fat content was determined gravimetrically.
- the main culture can be filtered through a filter paper (retention range 8-12 ⁇ m) at the end of the cultivation period and then lyophilized.
- the lyophilisate was then grindet with purified sand, mixed 3 times with approx. 75 ml hexane each and extracted with 40-120 bar pressure in a SpeedExtractor from Büchi. After removing the solvent with a rotary evaporator, the fat content was determined gravimetrically.
- FIG. 1 shows the fractions of fatty acids based on the total fatty acid content of the transesterified lipid extracts from different Conidiobolus strains; all fatty acids were determined by gas chromatography as fatty acid methyl esters. The fat content depending on the digestion method is shown in FIG. 2 .
- lipid extract obtained after extraction of the biomass was mixed with 10-100 ⁇ l of a lipase enzyme solution of “Lipase AY” of Amano (from Candida cylindracea ) having different concentrations, 100-190 ⁇ l phosphate buffer (200 mM, pH 7.6) and 100 ⁇ l water and incubated overnight in a thermoblock (Eppendorf) (45° C., 800 rpm).
- a 0.2 M Na 2 HPO 4 solution was prepared and mixed with a 0.2 M KH 2 PO 4 solution, so that a pH of 7.0 was obtained.
- the concentration ratios examined are shown in Table 4.
- ⁇ -Dioxygenase was carried out in accordance with literature references based on the method described in patent application WO 2012/025629 A1.
- 1 ⁇ L of the plasmid was pipetted to competent Escherichia coli cells [BL21(DE3), Novagen] for cell transformation. After a cooling phase (30 min on ice) the samples were heated for 2 min in a water bath (42° C.) and cooled again.
- 150 ⁇ L LB-Kanamycin medium (Table 5) was added and incubated for 1 h (37° C., 200 rpm, deflection 25 mm). The cell suspension was applied to pre-dried and acclimatized LB-kanamycin agar plates and incubated at 37° C. overnight.
- composition of the LB-Kanamycin medium Amount Components 10 g L ⁇ 1 Trypton 5 g L ⁇ 1 Yeast extract 10 g L ⁇ 1 NaCl 25 mg L ⁇ 1 Kanamycin (not autoclaved but added sterile filtered)
- this enzyme was expressed in E. coli cells [BL21(DE3)] on LB medium according to WO 2012 025629 A1.
- a 0.5 cm long smear was taken from the bacterial lawn using an inoculation loop, transferred to 3 mL LB kanamycin medium and incubated up to an OD 600 between 0.5-0.8 in a shaker (37° C., 150 rpm, deflection 25 mm).
- Isopropyl-D-thiogalactopyranoside (IPTG) was added (0.5 mM) and the sample incubated for another 16-18 h in the shaker (24° C., 150 rpm, deflection 25 mm).
- the sample was centrifuged (4,000 rpm, 3,724 ⁇ g, 10 min, 4° C.), the supernatant discarded and the cell pellet frozen ( ⁇ 20° C.).
- Frozen cell pellets produced as described above were taken up in phosphate puffer (200 mM, pH 7.6), washed in the same buffer, centrifuged and resuspended in 2 mL phosphate buffer to which 0.5% glucose monohydrate was added.
- phosphate puffer 200 mM, pH 7.6
- glucose monohydrate 0.5% glucose monohydrate was added.
- To 1 mL of the cell suspension 50 ⁇ L 12-methyltridecanoic acid (7.5 mg mL ⁇ 1 in DMSO) was added. The sample was incubated for 1 h at 37° C. (150 rpm, deflection 25 mm) and then extracted with 1 mL heptane. After drying the organic phase over sodium sulphate the sample was examined by gas chromatography.
- Example 4 The conversion was carried out in comparable manner as Example 4, in which a standard solution with 12-methyltridecanoic acid was used.
- a standard solution with 12-methyltridecanoic acid was used.
- 10 ⁇ l of the fat extract of the fungal culture from Example 2 was added, extracted as in Example 4 and then examined by gas chromatography.
- lipid extract of C. denaeosporus from Example 2 was weighed into a 10 mL vial and mixed with 200 ⁇ l lipase enzyme solution from Example 3, 200 ⁇ L phosphate buffer (200 mM, pH 7.6) and 200 ⁇ L water.
- 200 mg of the E. coli cells producing ⁇ -dioxygenase from Example 4 were washed with phosphate buffer (200 mM; pH 7.6) and resuspended in 4 mL phosphate buffer, which contained 0.5% glucose monohydrate, and added to the prepared 10 mL vial.
- the reaction preparation was incubated overnight (22 h, 24° C., 150 rpm, deflection 25 mm).
- the converted fat was extracted 3 times with 2 mL heptane each and the organic phases were combined. After dilution (1:50) the samples were analyzed by gas chromatography.
- the sample Prior to gas chromatographic analysis, the sample was mixed with 50 ⁇ L internal standard (750 mg L ⁇ 1 thymol). Alternatively, a reduction with an ⁇ -dioxygenase was carried out in a further experiment as shown in FIG. 6 .
- Protein expression of Nsp-CAR and MmFad9 was performed in recombinant E. coli BL21 (DE3) cells.
- 50 ml LB medium was inoculated with 30 ⁇ g kanamycin/ml from a master stock and incubated at 37° C. and 130 rpm until an OD 600 of about 0.6-0.8 was achieved.
- 1 mM IPTG was added for induction and the cultures were continued to grow overnight at room temperature.
- the biomass thus obtained was centrifuged at 10,000 rpm for 10 minutes and the cell pellet was stored at ⁇ 20° C. until further use.
- the pellets stored at ⁇ 20′C were thawed and resuspended with 4 ml B-PER reagent per g pellet. After adding 50 ⁇ g lysozyme/ml and 2.5 U DNase/ml, the samples were incubated for 15 minutes at room temperature, then centrifuged for 10 minutes at 10,000 rpm and 4° C. to obtain the total protein extract. After subsequent purification of the enzymes using HisPur Ni-NTA columns, the protein concentrations of the purified enzymes were determined according to Bradford (9.92 mg/ml for Nsp-CAR and 26.43 mg/ml for MmFad9).
- FIG. 4 1 shows the mass spectra of the standard of 12-methyltridecanal (right part) and after reduction with Nsp-CAR (left part).
- FIG. 4 left: Mass spectrum of 12-methyltridecanal generated from homologous acid by reduction using CAR; right: mass spectrum of the standard of 12-methytridecanal
- E. coli cells expressing reductase and the auxiliary enzyme were initially grown to cultures so that they reached an OD of about 2.
- the biotransformation preparations were then prepared in such a way that for each preparation each reductase was present once with respectively without auxiliary protein as well as a preparation each as a positive control, in which 100 ⁇ l of a benzoic acid solution was added to each reductase. In addition to the volumes of about 1 ml each, which were required for the expressing E.
- FIG. 5 top: MS/MS chromatogram (TIC, scan in Q1) of the blank value (heat-inactivated CAR) of the enzymatic conversion of the lipid extract; center: Reaction of the lipid extract without addition of lipase; bottom: Conversion of the lipid extract with lipase and CAR
- 1 mL was taken from a cryopreserve of Nocardia iowensis (DSM 45197) and transferred to a 100 ml flask containing 10 ml LB medium and 0.05% Tween 80. The flask was cultivated for approx. 72 h at 28° C. and 130 rpm with a shaking amplitude of 50 mm. Afterwards, 1 ml was taken from the well-grown culture and transferred to a 100 ml Erlenmeyer flask with also 10 ml of the same fresh medium. Analogous to T. Li et. al. ( J Bacteriol.
- the previously obtained fat extracts were divided into two qualities: those with a fat content of less than 1 g/L and those with higher contents.
- the conversion occurred in two steps, first as reduction with the help of lithium alanate and then as oxidation with TEMPO/KBr/NaOCl.
- the reaction conditions are given in Table 10 below:
- FIGS. 7A and 7B show the composition of the resulting alcohols before and after ball tube distillation for the two preparations designated 078 ( ⁇ 1 g/L) and 083 (>1 g/L).
- the obtained aldehyde fractions (#081 and #085) were pooled and again distilled in the ball tube. 19.6 g (GC purity 44.8%) corresponding to a yield of 77% based on the amount of 12-methyltridecanoic acid or the corresponding glycerides were determined. The results are shown in FIG. 8A .
- the resulting distillate was subjected to further purification in the split tube (Spaltrohr) (72-83° C., 0.5 mbar).
- Various fractions with purities of more than 95% were obtained ( FIG. 8B ).
- the compositions are shown in Table 11.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Seasonings (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
- (a) providing a culture of one or more fungi of the genus Conidiobolus and producing biomass containing branched carboxylic acids in free and/or bound form;
- (b) extracting the biomass from step (a) to produce a first intermediate containing free and/or bound carboxylic acids;
- (c) optionally chemically, enzymatically or microbially hydrolyzing the bound carboxylic acids from the first intermediate;
- (d) treating the first intermediate with a reducing agent of a chemical nature to convert the free and/or bound carboxylic acids into the corresponding alcohols and optionally separating one or more alcohols from interfering by-products and producing the chemically produced second intermediate containing these alcohols as a mixture or in enriched form;
- (e) treating the first intermediate with a reducing agent of a biological nature to convert the free and/or bound carboxylic acids into the corresponding aldehydes having the same number of carbon atoms compared to the free and/or bound carboxylic acids or into the corresponding aldehydes having a reduced number of carbon atoms by one compared to the free and/or bound carboxylic acids and producing the biologically produced second intermediate containing these aldehydes;
- (f) treating the chemically produced second intermediate with an oxidizing agent of a chemical nature to convert the free and/or bound alcohols into the corresponding aldehydes; and optionally
- (g) removing interfering by-components from the fractions obtainable after step(s) (d) and/or (e) and/or (f).
Description
- The invention is in the field of flavoring substances and relates to a new method for the production of branched aldehydes, in particular 12-methyltridecanal.
- Branched aldehydes are interesting odorous and flavoring substances that are prevalent in nature. 12-methyltridecanal in particular is an important building block for authentically tasting bovine flavors and can currently only be commercially produced by chemical synthesis (cf. Kerscher, et. al., J. Agric. Food Chem. 48(6), p. 2387-2390, 2000).
- Based on the manufacturing process, it can therefore not be declared natural in the sense of European flavor legislation. On the other hand, there is a special interest on the market in flavors that can be declared not only as nature-identical, but explicitly as natural; this declaration option also represents an important purchase and price argument.
- The objective of the present invention was therefore to provide a method for the production of branched aldehydes in general and 12-methyltridecanal in particular by biotechnological and therefore natural means.
- A first object of the invention relates to a method for the production of branched aldehydes, preferably those comprising 12 to 18 carbon atoms and/or having a methyl branching, in particular 12-methyltridecanal, 12-methyltetradecanal, 14-methylpentadecanal, 16-methyloctadecanal or mixtures thereof, the method comprising the following steps:
- (a) providing a culture of one or more fungi of the genus Conidiobolus and producing biomass containing branched carboxylic acids in free and/or bound form;
- (b) extracting the biomass from step (a) to produce a first intermediate containing free and/or bound carboxylic acids;
- (c) optionally hydrolyzing the bound carboxylic acids from the first intermediate chemically, enzymatically or microbially;
- (d) treating the first intermediate with a reducing agent of a chemical nature to convert the free and/or bound carboxylic acids into the corresponding alcohols and optionally separating one or more alcohols from interfering by-components and producing the chemically produced second intermediate containing these alcohols as a mixture or in enriched form;
- (e) treating the first intermediate with a reducing agent of a biological nature to convert the free and/or bound carboxylic acids into the corresponding aldehydes having the same number of carbon atoms as the free and/or bound carboxylic acids or into the corresponding aldehydes having a reduced number of carbon atoms by one compared to the free and/or bound carboxylic acids and producing the biologically produced second intermediate containing these aldehydes;
- (f) treating the chemically produced second intermediate with an oxidizing agent of a chemical nature to convert the free and/or bound alcohols into the corresponding aldehydes; and optionally
- (g) removing interfering by-components from the fractions obtainable after steps (d) and/or (e) and/or (f).
- Surprisingly, it was found that fungal genus Conidiobolus that has previously received little attention has a fatty acid pattern that makes this microorganism appear as a suitable starting material for the production of branched aldehydes in general and 12-methyltridecanal in particular. The final products can be obtained in sufficient yield and high purity by an optimized sequence of treatment with biological and/or chemical reducing agents and chemical oxidizing agents. The oxidized products are mixtures of branched aldehydes, which also contain linear species. These mixtures can be used directly, but it is also possible to obtain the highly desired product 12-methyltridecanal by targeted processing.
- Organisms
- The organisms which, due to the fatty acid spectrum they produce, serve as suitable bioreactors for the production of branched fatty acids of the desired chain length belong to a special genus of fungi, namely Conidiobolus, which mainly comprises saprobions that degrade dead plant material or live in the soil. The two preferred species C. denaeosporus and C. heterosporus are described in connection with decomposition of leaves of the Chinese elm (Ulmus parvifolia). Taxonomically, the two genera are classified as follows:
- Kingdom: Fungi
- Subphylum: Entomophthoromycotina
- Order: Entomophthorales
- Family: Ancylistaceae
- Overviews of these organisms were published by D. Tyrrell in the years 1968 to 1976: “The fatty acid composition of some Entomophthoraceae. II. the occurrence of branched-chain fatty acids in Condiobolus denaesporus” Lipids 3 (4), p. 368-372 (1968); “Fatty acid composition of some Entomophthoraceae. III” Can J Microbiol 17 (8), p. 1115-1118 (1971); and “The fatty acid composition of some Entomophthoraceae. IV. The occurrence of branched-chain fatty acids in Conidiobolus species”. Can J Microbiol 22 (7), pp. 1058-1060 (1976).
- Cultivation
- Fungal mycelium/mycelia can be cultivated in a known manner using a suitable nutrient medium in solid or liquid culture. Agar plates produced with a nutrient solution of malt and yeast extract have proved their worth.
- Extraction
- After the cultures have formed a sufficient amount of biomass, the biomass, if necessary, is separated from the nutrient solution for example by centrifuging, filtration or other suitable separation methods. This is followed by extraction of carboxylic acids in their free or bound form. Solid cultures, such as those obtained after freeze-drying, can be extracted directly.
- Further, insofar as reference has been made to extracts, they may be produced in a manner known per se, i.e. for example by aqueous, alcoholic or aqueous-alcoholic extraction of the cultures themselves or dry mass obtained therefrom. All conventional extraction methods are suitable, e.g. maceration, remaceration, digestion, motion maceration, vortex extraction, ultrasonic extraction, countercurrent extraction, percolation, repercolation, evacolation (extraction under reduced pressure), diacolation or solid-liquid extraction under continuous backflow. The percolation method is advantageous for large-scale use. Organic solvents, water (preferably hot water at a temperature above 80° C. and in particular above 95° C.) or mixtures of organic solvents and water, in particular low-molecular weight alcohols with more or less high water contents, may be used as solvents for carrying out the extractions.
- Extraction with methanol, ethanol, pentane, hexane, heptane, acetone, propylene glycols, polyethylene glycols, ethyl acetate and mixtures thereof and their aqueous mixtures is particularly preferred. The extraction usually takes place at 20 to 100° C., preferably at 30 to 90° C., in particular at 60 to 80° C. In a preferred embodiment, extraction is carried out in an inert gas atmosphere to avoid oxidation of the active ingredients of the extract. This is particularly important for extractions at temperatures above 40° C. The durations of extraction are set by the skilled person depending on the starting material, the extraction method, the extraction temperature, the ratio of solvent to raw material and others.
- After extraction, the raw extracts obtained may undergo further conventional steps, such as purification, concentration and/or discoloration. If desired, the extracts formed in this way can, for example, be subjected to a selective separation of individual undesirable ingredients. The extraction can occur to any degree of extraction, but is usually carried out until exhaustion. Typical yields (=amount of dry substance of the extract based on the amount of raw material used) in the extraction of the dry biomass are in the range of 3 to 15, in particular 6 to 10% by weight. The present invention encompasses the finding that the extraction conditions and the yields of the final extracts can be selected by the skilled person according to any desired field of application. These extracts, which usually contain active substance contents (=solids contents) in the range of 0.5 to 10% by weight, can be used as such, but it is also possible to completely remove the solvent by drying, especially by spray or freeze drying.
- The extracts, which are hereinafter also referred to as the first intermediate, may be present as aqueous preparations and/or preparations dissolved in organic solvents as well as spray-dried or freeze-dried, anhydrous solids. In this context, aliphatic or branched alcohols with 1 to 6 carbon atoms (e.g. ethanol, 2-methoxy-2-methylpropane), ketones (e.g. acetone), halogenated hydrocarbons (e.g. chloroform or methylene chloride), lower esters or polyols (e.g. glycerol or glycols) are possible organic solvents.
- Hydrolysis
- If necessary, the extracts can be subjected to hydrolysis before the reduction to release the fraction of fatty acids bound as glyceride. The hydrolysis can occur chemically, but a biological alternative is preferable. This alternative can occur microbially, i.e. using living microorganisms, as well as enzymatically, since these hydrolyses are gentler and also offer regulatory advantages. Hydrolases from Candida, in particular Candida rugosa or Candida cylindracea are suitable for the biological alternative.
- Reduction
- After extraction, a first intermediate of a mixture of different linear and branched, saturated and unsaturated fatty acids, which can also be bound as glycerides, is obtained. Such a mixture may include, for example, the following representatives: tridecanoic acid, 12-methyltridecanoic acid, tetradecanoic acid, 12-methyltetradecanoic acid, pentadecanoic acid, 14-methylpentadecanoic acid, hexadecanoic acid, hexadecen(9)acid, 15-methylhexadecanoic acid, heptadecanoic acid, elaidic acid, oleic acid and arachidonic acid.
- The mixture of fatty acids or fatty acid glycerides is now subjected to a reduction. Like hydrolysis, this can be done chemically, but preferably a biological alternative is chosen, as this then leads to a production method that is considered natural in the sense of European flavoring legislation. The preferred biological alternative can be carried out with enzymes as well as with cell lysates, cell extracts or whole cells that have the enzymatic activity required for the reduction. After this step, the reaction products can be extracted from the reaction mixture using extraction methods known to the skilled person. After removal of the extractant, the concentrate obtained can then be taken up in the desired concentration with the solvent of choice.
- The chemical reduction of the mixture of fatty acids respectively fatty acid glycerides can be carried out with sodium borohydride, but lithium alanate should preferably be used. In the course of this conversion, the acid or ester functions are reduced to hydroxyl groups; the second, in this case chemical intermediate, thus completely or at least predominantly contains the corresponding alcohols of the linear and branched carboxylic acids. Within reaction times of about 3 hours, yields of 85% to 95% of the theoretical yield are achieved, which can be increased if the reduction is followed by another distillation step, for example a ball tube (bulb to bulb; Kugelrohr) distillation. Based on the amount of the particularly desired product 12-methytridecanol as a reduction product of 12-methytridecanoic acid, the yield is around 25 to 35% based on the total amount of reduction products. As a side reaction, the reduction also results in the saturation of unsaturated species contained in the mixture.
- Reduction methods using hydrides belong to the standard reactions of a preparative organic chemists and represent textbook knowledge that therefore requires no further comprehensive explanation. The reduction is preferably carried out at temperatures in the range of 15 to 25° C., whereby, as expected, a temporary temperature increase occurs at the beginning of the reaction.
- Oxidation
- The second chemical intermediate serves as a starting material for the subsequent oxidation, during which the alcohols are converted into the corresponding aldehydes. TEMPO is preferably used as an oxidizing agent. TEMPO stands for 2,2,6,6-tetramethylpiperidinyloxyl.
- Although this radical is not thermodynamically stable, its comparatively high persistence is due to substituents that influence the service life through steric effects. The substituents are located in the vicinity of the radical electron, so that it has an average life of one minute in an oxygen-free solution. The use of TEMPO together with a suitable co-oxidant for the two-phase oxidation of alcohols is described by Anelli et al. in J Organic Chemistry. 52, p. 2559-2562 (1987) (“Anelli oxidation”) and used for example in Organic Syntheses, Coll. Vol. 8, p. 367 (1993); Vol. 69 p. 212 (1990).
- For the purposes of this invention, it has proven advantageous to use TEMPO in combination with two co-oxidants, namely an alkali bromide and an alkali hypochlorite. A combination of potassium bromide and sodium hypochlorite is preferred here. A molar ratio of TEMPO, alkali bromide and alkali hypochlorite of about 1:(2 to 10):(10 to 40), particularly about 1:(2 to 10):(10 to 40) and more particularly of about 1:(4 to 8):(15 to 30) has proven its worth. Since the reaction is highly exothermic, it is recommended to keep the reaction temperature in the range of about −5 to +10° C.
- Under these conditions, yields in the range of 85 to 97% based on the alcohols used are obtained. In relation to the preferred target product 12-methyltridecanal the yield is 30 to 35%, whereby a purity of approx. 95% is achieved.
- Also in the case of the oxidation, purity can be further improved by subjecting the products to a distillation step, such as a ball tube (Kugelrohr) distillation or by means of a split tube (Spaltrohr).
- When selecting an enzymatic process for the biological reduction, enzymes such as commercially available aldehyde dehydrogenases (ALDH, EC 1.2.1.x) and carboxylic acid reductases, e.g. from Norcardia sp. (Aimin He, Tao Li, Lacy Daniels, Ian Fotheringham, John P. N. Rosazza Appl Environ Microbiol. 2004 March; 70(3): 18744881) and/or Mycobacterium marinum (M. Kalim Akhtar, Nicholas J. Turner, Patrik R. Jones Proc Natl Acad Sci USA. 2013 Jan. 2; 110(1): 87-92), can be used. Carboxylic acid reductases can be obtained by homologous expression in Nocardia sp. or Mycobacterium sp., in particular Nocardia iowensis, as well as heterologously by recombinant expression in suitable host organisms in particular E. coli, but preferably also by cultivation of Nocardia iowensis, which as so-called wild-type strain also expresses the enzymes. For the actual conversion it is irrelevant whether the enzymes are present in purified form, are only partially concentrated, or are present in a cell raw extract or in native or recombinant cells.
- A further object of the invention concerns a concentrate of methyl-branched aldehydes having 12 to 18 carbon atoms, obtainable by the method described above, which concentrate can contain the methyl-branched aldehydes in amounts of 10 to 100, preferably 25 to 95 and in particular about 40 to about 60% by weight. The concentrates can be obtained directly on the basis of the chemically formed second intermediate (f), the biologically formed second intermediate (e) or the completely or partially purified products (g) by suitable, preferably gentle drying methods, such as spray drying or freeze drying. The difference to 100% by weight may be due to carriers and/or other flavoring substances. Alternatively, concentrated aqueous solutions can also be sold.
- A final object of the present invention concerns the use of the concentrates or alternatively the products directly obtained by the process as described above as flavoring components, in particular as flavoring components for foodstuffs, in order to give them a beef flavor.
- Used Fungal Strains
- Table 1 below gives an overview of the fungi examined, their exact designation, number of the strain collection (CBS number), the isolation substrate and the isolation site.
-
TABLE 1 Fungi assessed CBS Isolation substrate and Abbrevia- Organism number site tion Conidiobolus 137.57 decomposed leaves of CDE denaeosporus Ulmus parvi-folia, (US) Conidiobolus 333.74 agricultural soil (NL) CHET-N heterosporus Conidiobolus 138.57 Forest leaf litter (US) CHET-U heterosporus - Cultivation on Solid Medium
- The fungal strains were cultivated on malt extract agar plates. To produce the plates, the media components were weighed (Table 2), filled up with distilled water to 1 litre and autoclaved (120° C., 20 min). A 1 cm2 mycelium-covered piece of agar from the vital peripheral area of a plated culture was placed in the middle of the agar plates, the petri dishes were sealed with parafilm and then incubated with or preferably without light at 24° C. in the incubator. After about 5 days (CHET-N) and 14 days (CHET-U and CDE), pre-cultures were inoculated from the plates that were about three-quarters overgrown. In parallel, further agar plates for the next pre-cultures were inoculated according to the same principle. Table 2 shows the composition of the malt extract-yeast extract-agar for the cultivation of Conidiobolus strains:
-
TABLE 2 Composition agar Medium component Concentration [g L−1] Agar 15 Malt extract 30 Yeast extract 3 - For the submerged pre-cultures a complex nutrient medium was used, which is shown in Table 3:
-
TABLE 3 Composition of the standard complex medium Medium component Concentration [g L−1] Malt extract 30 Yeast extract 3 - For cultivation in submerged culture, a 1 cm2 agar piece of the outer mycelium was transferred under sterile conditions to a 250 mL Erlenmeyer flask filled with 100 mL culture medium and treated with an ultraturrax dispersing rod (30 s, 9,800 rpm). The pre-cultures were incubated at 24° C. in an incubator (Multitron, Infors, Einsbach; 24° C., 150 rpm,
deflection 25 mm) with no light. - Main culture: For cultivation of the main cultures, the pre-culture was treated under sterile conditions with an ultraturrax dispersing rod (30 s, 9,800 rpm). 20 mL of this suspension was added to 200 mL fresh culture medium in a 500 mL Erlenmeyer flask. Cultivation took place at 24° C. with exclusion of light in an incubation shaker (24° C., 150 rpm,
deflection 25 mm). - The main culture was filtered through a filter paper (range of retention 8-12 μm) at the end of the cultivation period. The residue was mixed with about 200 mL 4 M HCl, heated to boiling point and digested for 30 min in the boiling heat. The solution was then filtered through a pleated filter while still hot and washed with hot water until the wash water was neutral. After drying for 1-2 h in a drying cabinet (100° C.), the filter paper was extracted for 4 h with petroleum gasoline (boiling range 40-60° C.) using a Soxhlet apparatus. After removing the solvent with a rotary evaporator, the fat content was determined gravimetrically.
- Alternatively, the main culture can be filtered through a filter paper (retention range 8-12 μm) at the end of the cultivation period and then lyophilized. The lyophilisate was then grindet with purified sand, mixed 3 times with approx. 75 ml hexane each and extracted with 40-120 bar pressure in a SpeedExtractor from Büchi. After removing the solvent with a rotary evaporator, the fat content was determined gravimetrically.
- Various approaches were investigated to identify the most suitable processing method for the fermentatively produced biomass. Lyophilization with subsequent fine-grinding was identified as the most suitable method.
FIG. 1 shows the fractions of fatty acids based on the total fatty acid content of the transesterified lipid extracts from different Conidiobolus strains; all fatty acids were determined by gas chromatography as fatty acid methyl esters. The fat content depending on the digestion method is shown inFIG. 2 . - About 50 mg of the lipid extract obtained after extraction of the biomass was mixed with 10-100 μl of a lipase enzyme solution of “Lipase AY” of Amano (from Candida cylindracea) having different concentrations, 100-190 μl phosphate buffer (200 mM, pH 7.6) and 100 μl water and incubated overnight in a thermoblock (Eppendorf) (45° C., 800 rpm). To prepare the buffer, a 0.2 M Na2HPO4 solution was prepared and mixed with a 0.2 M KH2PO4 solution, so that a pH of 7.0 was obtained. The concentration ratios examined are shown in Table 4. Subsequently, the samples were shaken out with 2 mL hexane and the upper (organic) phase was filled into glass vials to determine the fatty acid distribution by GC (without previous methylation) (
FIG. 3 ). No free fatty acids were present in the untreated fat fraction. By hydrolysis with the lipase fatty acids were released, strongly dependent on the amount of enzyme used (with 3 U enzyme only very little is released, but with 30 U substantially more). A total of 14% 12-methyltridecanoic acid was found. In a repetition of the experiments with 100 U and 300 U enzymes a total of 25% by weight of 12-methyltridecanoic acid was determined. -
TABLE 4 Reaction preparations for hydrolysis of the fat extract A B C D Fat fraction 50 mg 50.4 mg 51 mg 51.3 mg Lipase solution 10 μl 100 μl (10 mg/ml) Lipase solution 20 μl 100 μl (100 mg/ml) Amount of lipase 3 30 100 300 (U Lipase) Phosphate buffer 190 μl 100 μl 180 μl 100 μl (200 mM), pH 7 H2O 100 μl 100 μl 100 μl 100 μl - Conversion of 12-Methyltridecanoic Acid with α-Dioxygenase
- The production of α-Dioxygenase was carried out in accordance with literature references based on the method described in patent application WO 2012/025629 A1. 1 μL of the plasmid was pipetted to competent Escherichia coli cells [BL21(DE3), Novagen] for cell transformation. After a cooling phase (30 min on ice) the samples were heated for 2 min in a water bath (42° C.) and cooled again. For cultivation 150 μL LB-Kanamycin medium (Table 5) was added and incubated for 1 h (37° C., 200 rpm,
deflection 25 mm). The cell suspension was applied to pre-dried and acclimatized LB-kanamycin agar plates and incubated at 37° C. overnight. -
TABLE 5 Composition of the LB-Kanamycin medium Amount Components 10 g L−1 Trypton 5 g L−1 Yeast extract 10 g L−1 NaCl 25 mg L−1 Kanamycin (not autoclaved but added sterile filtered) - For the conversions using α-dioxygenase, this enzyme was expressed in E. coli cells [BL21(DE3)] on LB medium according to WO 2012 025629 A1. A 0.5 cm long smear was taken from the bacterial lawn using an inoculation loop, transferred to 3 mL LB kanamycin medium and incubated up to an OD600 between 0.5-0.8 in a shaker (37° C., 150 rpm,
deflection 25 mm). Isopropyl-D-thiogalactopyranoside (IPTG) was added (0.5 mM) and the sample incubated for another 16-18 h in the shaker (24° C., 150 rpm,deflection 25 mm). The sample was centrifuged (4,000 rpm, 3,724×g, 10 min, 4° C.), the supernatant discarded and the cell pellet frozen (−20° C.). - Frozen cell pellets produced as described above were taken up in phosphate puffer (200 mM, pH 7.6), washed in the same buffer, centrifuged and resuspended in 2 mL phosphate buffer to which 0.5% glucose monohydrate was added. To 1 mL of the
cell suspension 50 μL 12-methyltridecanoic acid (7.5 mg mL−1 in DMSO) was added. The sample was incubated for 1 h at 37° C. (150 rpm,deflection 25 mm) and then extracted with 1 mL heptane. After drying the organic phase over sodium sulphate the sample was examined by gas chromatography. - Conversion of the Lipid Extract with α-Dioxygenase
- The conversion was carried out in comparable manner as Example 4, in which a standard solution with 12-methyltridecanoic acid was used. To 2 ml of the E. coli cells resuspended in phosphate buffer, 10 μl of the fat extract of the fungal culture from Example 2 was added, extracted as in Example 4 and then examined by gas chromatography.
- Simultaneous Conversion with Lipase and with α-Dioxygenase
- 145 mg of a lipid extract of C. denaeosporus from Example 2 was weighed into a 10 mL vial and mixed with 200 μl lipase enzyme solution from Example 3, 200 μL phosphate buffer (200 mM, pH 7.6) and 200 μL water. 200 mg of the E. coli cells producing α-dioxygenase from Example 4 were washed with phosphate buffer (200 mM; pH 7.6) and resuspended in 4 mL phosphate buffer, which contained 0.5% glucose monohydrate, and added to the prepared 10 mL vial. The reaction preparation was incubated overnight (22 h, 24° C., 150 rpm,
deflection 25 mm). The converted fat was extracted 3 times with 2 mL heptane each and the organic phases were combined. After dilution (1:50) the samples were analyzed by gas chromatography. - Conversion with Aldehyde Dehydrogenase (ALDH)
- For conversion with aldehyde dehydrogenase (ALDH from Saccharomyces cerevisiae, 10.5 U mg−1 protein), about 15 mg 12-methyltridecanoic acid was emulsified with 500 μL Triton-X-100 solution (2.2 g L−1). The conversion was carried out in accordance with Sigma Quality Control Test1 based on Bostian et al., (1978) Biochemical Journal 173, 773-786. After enzyme addition the samples were incubated in a shaker (30 min, 24° C., 150 rpm,
deflection 25 mm) and then shaken out twice with 1 mL pentane/diethyl ether (1:1.12, v/v) each. Prior to gas chromatographic analysis, the sample was mixed with 50 μL internal standard (750 mg L−1 thymol). Alternatively, a reduction with an α-dioxygenase was carried out in a further experiment as shown inFIG. 6 . - Reduction of 12-methlytridecanoic Acid with Carboxylic Acid Reductases
- Protein expression of Nsp-CAR and MmFad9 was performed in recombinant E. coli BL21 (DE3) cells. For this, 50 ml LB medium was inoculated with 30 μg kanamycin/ml from a master stock and incubated at 37° C. and 130 rpm until an OD600 of about 0.6-0.8 was achieved. When this value was reached, 1 mM IPTG was added for induction and the cultures were continued to grow overnight at room temperature. The biomass thus obtained was centrifuged at 10,000 rpm for 10 minutes and the cell pellet was stored at −20° C. until further use.
- To recover protein, the pellets stored at −20′C were thawed and resuspended with 4 ml B-PER reagent per g pellet. After adding 50 μg lysozyme/ml and 2.5 U DNase/ml, the samples were incubated for 15 minutes at room temperature, then centrifuged for 10 minutes at 10,000 rpm and 4° C. to obtain the total protein extract. After subsequent purification of the enzymes using HisPur Ni-NTA columns, the protein concentrations of the purified enzymes were determined according to Bradford (9.92 mg/ml for Nsp-CAR and 26.43 mg/ml for MmFad9).
- The enzyme assay was performed using 50 mM Tris-HCl buffer (pH=7.5), 20 mM substrate, 10 mM ATP, 10 mM NADPH, 100 mM MgCl2 and 1 mg of the purified enzyme so that the total volume was 1.1 ml. After an incubation period of 24 h at room temperature, the aqueous mixture was shaken out 3 times each with 1:1 hexane and the upper, organic phase was transferred to a GC vial. The results obtained are summarized in Table 6.
FIG. 4 1 shows the mass spectra of the standard of 12-methyltridecanal (right part) and after reduction with Nsp-CAR (left part). 1FIG. 4 : left: Mass spectrum of 12-methyltridecanal generated from homologous acid by reduction using CAR; right: mass spectrum of the standard of 12-methytridecanal -
TABLE 6 12-methyltridecanoic acid and 12-methyltridecanal contents of the enzymatic conversions 12-Methyltridecanal 12-Methyltridecanoic acid [μg/ml] [μg/ml] Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 MmFad9; 1st extraction 1.38 1.755 1.98 3105 3382.5 3333 MmFad9; 2nd extraction 0.2125 0.4 0.3625 153.75 192.5 197.5 MmFad9; 3rd extraction — — — 12 20 22 Nsp-CAR; 1st extraction 1.475 1.7125 1.8625 2850 2975 2975 Nsp-CAR; 2nd extraction 0.3 0.475 0.3 153.75 162.5 160 Nsp-CAR; 3rd extraction — — — 20 22 23 - Reduction of 12-Methyltridecanoic Acid in a Biotransformation (with Living E. coli BL21 Cells that can Express Either Nsp-CAR or MmFad9)
- By the fact that Akhtar et al. (P Natl Acad Sci USA 2013; 110: 87-92) described that the catalytic activity of reductases can be positively influenced by the presence of the protein BsSfp, a corresponding E. coli strain was grown in parallel in LB medium at 37° C. and 150 rpm overnight and added to the biotransformation preparation.
- E. coli cells expressing reductase and the auxiliary enzyme were initially grown to cultures so that they reached an OD of about 2. The biotransformation preparations were then prepared in such a way that for each preparation each reductase was present once with respectively without auxiliary protein as well as a preparation each as a positive control, in which 100 μl of a benzoic acid solution was added to each reductase. In addition to the volumes of about 1 ml each, which were required for the expressing E. coli strains, 5 μl IPTG (final concentration in the
preparation 1 mM), 100 μl of an ethanolic 12-methyltridecanoic acid solution (final concentration in thepreparation 2 mM) and about 3 to 4 ml fresh LB medium were added to each preparation, so that after completion the preparations had an OD of about 0.5 in a final volume of 5 ml. The preparations thus prepared were cultivated in duplicates for 24 h at room temperature at a shake frequency of 150 rpm. Subsequently, each preparation was extracted 2 times with hexane in a ratio of 1:1. The results from the biotransformations are listed in Table 7. -
TABLE 7 Results of biotransformations for the formation of 12-methyltridecanal 12-Methyltridecanal 12-Methyltridecanoic acid 12-Methyltridecanol Enzyme μg/ml μg/ml μg/ml Nsp-CAR 0.77 0.75 5.97 5.80 24.7 22.5 Nsp-CAR + BsSfp 0.58 0.51 13.09 12.68 11.1 10.5 MmFad9 0.31 0.31 58.32 63.51 2.5 2.8 MmFad9 + BsSfp 0.28 0.33 62.33 64.93 0.9 0.9 - Hydrolysis of a Fat Fraction of Conidiobolus heterosporus Using Lipase and Subsequent Conversion with the Carboxylic Acid Reductases MmFad9 Respectively Nsp-CAR to Form 12-methyltridecanal
- Hydrolysis was performed with pooled lipid extracts, as they can be obtained following the description of Example 2, in accordance with the description of Example 3. 150 mg of the thus hydrolyzed fat extract were mixed with 1010 μl of a reaction mixture as shown in Table 8. MS/MS analyses, shown in
FIG. 5 2, showed that 12-methyltridecanal could be identified with both hydrolyzed and non-hydrolyzed fat extracts. 2FIG. 5 : top: MS/MS chromatogram (TIC, scan in Q1) of the blank value (heat-inactivated CAR) of the enzymatic conversion of the lipid extract; center: Reaction of the lipid extract without addition of lipase; bottom: Conversion of the lipid extract with lipase and CAR -
TABLE 8 Composition of the reaction mixture for reducing hydrolyzed fat extract Substance Amount used CAR protein solution from Example 8 900 μL ATP disodium salt 1 mM NADPH tetranodium salt 1 mM MgCl2 * 6 H2O 10 mM final volume 1.010 ml - Reduction of a Fat Extract of Conidiobolus heterosporus with Living Cells of Nocardia iowensis for the Production of 12-methytridecanal
- 1 mL was taken from a cryopreserve of Nocardia iowensis (DSM 45197) and transferred to a 100 ml flask containing 10 ml LB medium and 0.05
% Tween 80. The flask was cultivated for approx. 72 h at 28° C. and 130 rpm with a shaking amplitude of 50 mm. Afterwards, 1 ml was taken from the well-grown culture and transferred to a 100 ml Erlenmeyer flask with also 10 ml of the same fresh medium. Analogous to T. Li et. al. (J Bacteriol. June 1997; 179(11): 3482-3487) 5 mg benzoic acid/ml was added for induction and the flask was incubated for another 24 h at 28° C. and 130 rpm. Subsequently, biocatalyses were carried out according to the compilation of Table 9. After the induction phase, the substrate was added and the preparations were incubated for another 24 h at 28° C. and 150 rpm. The samples were then extracted 2:1 with hexane, the organic phase was transferred to a sample container and examined by gas chromatography for 12-methyltridecanoic acid and 12-methyltridecanal. The results are summarized in Table 9: -
TABLE 9 Experimental results from the biocatalysis tests 12-Methyl- 12-Methyl- tridecanal tridecanoic acid Substrate [μg/ml] [μg/ml] Lipase-hydrolyzed fat fraction of C. 0.39 8.4 heteropsorus (from Example 3) Fat fraction of C. heterosporus — 6.9 dissolved in DMSO without prior hydrolysis (from Example 2) 12-Methyltridecanoic acid — 107.0 Fat fraction of C. heterosporus 0.60 42.7 without prior hydrolysis - Chemical Conversion of Fat Extracts from C. Heterosporus to 12-methltridecanal
- The previously obtained fat extracts were divided into two qualities: those with a fat content of less than 1 g/L and those with higher contents. The conversion occurred in two steps, first as reduction with the help of lithium alanate and then as oxidation with TEMPO/KBr/NaOCl. The reaction conditions are given in Table 10 below:
-
TABLE 10 Reaction conditions* Purity by GC-MS [%] after ball tube Prepara- Yield (Kugelrohr) tion Reaction conditions [g] [%] direct distillation 078 LiAlH4 (0.7 eq.) RT, 3 h 17.3 89 33.1 37.4 083 LiAlH4 (0.7 eq.) RT, 3 h 10.7 93 30.3 37.2 081 KBr (0.28 eq.), TEMPO 16.0 87 36.9 44.8 (0.067 eq.), NaClO (1.2 eq.), 0° C., 4 h 085 KBr (0.30 eq.), TEMPO 34.2 (0.073 eq.), NaClO (1.2 eq.), 0° C., 3 h *078: 27.4 g of <1 g/L, 083: 17.8 g of >1 g/L, 081: <1, 085: >1 - The reduction with lithium alanate yielded 89% for the <1 g/L experiment and 93% for the >1 g/L experiment (in each case after ball tube distillation) based on the content of 12-methyltridecanoic acid or the corresponding glycerides. The proportion of double bonds was reduced from 22% to 6% and from 29% to 22%, respectively.
FIGS. 7A and 7B show the composition of the resulting alcohols before and after ball tube distillation for the two preparations designated 078 (<1 g/L) and 083 (>1 g/L). - The subsequent oxidation with TEMPO gave an aldehyde yield of 97% (081) for the case <1 g/L and 94% for the case >1 g/L (085) based on the content of 12-methyltridecanoic acid respectively the corresponding glycerides (in each case before ball tube distillation). The results are shown in
FIG. 7C . - The obtained aldehyde fractions (#081 and #085) were pooled and again distilled in the ball tube. 19.6 g (GC purity 44.8%) corresponding to a yield of 77% based on the amount of 12-methyltridecanoic acid or the corresponding glycerides were determined. The results are shown in
FIG. 8A . The resulting distillate was subjected to further purification in the split tube (Spaltrohr) (72-83° C., 0.5 mbar). Various fractions with purities of more than 95% were obtained (FIG. 8B ). The compositions are shown in Table 11. -
TABLE 11 Purity of the fractions from split tube distillation Purity 12-MTD (GC) Amount [g] 085 KD1 Fr1 44.8% 19.6 Split tube Fr1 — 1.1 Split tube Fr2 0.1% 0.1 Split tube Fr3 2.9% 1.1 Split tube Fr4 72.1% 1.1 Split tube Fr5 93.2% 1.1 Split tube Fr6 90.6% 0.2 Split tube Fr7 94.1% 0.4 Split tube Fr8 92.9% 0.3 Split tube Fr9 96.2% 1.0 Split tube Fr10 96.2% 0.4 Split tube Fr11 97.1% 0.5 Split tube Fr12 94.7% 0.2 Residue 13.3% 12.2 - The total yield of oxidation of the two pooled qualities from fractions 5 to 12 (4.1 g, purity 94.7%) was 38% based on the alcohol mixtures (17 g with 37.4% purity and 10.5 g with 37.2% purity).
- The yield of 12-methytridecanal based on both steps (reduction and oxidation of 27.4 g<1 g/L fat extract with 30.4 GC-% 12-methyltric acid methyl ester and 17.8 g>1 g/L with 27.1 GC-% ester) was 34% based on the fractions 5 to 12 (4.1 g, purity 94.7%).
- Based on the 95% purity of fractions 5, 7 and 9 to 12 (3.6 g), the yield after both steps was 30%.
Claims (21)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/EP2015/077992 WO2017088937A1 (en) | 2015-11-28 | 2015-11-28 | Method for producing branched aldehydes |
Publications (2)
Publication Number | Publication Date |
---|---|
US20190367954A1 true US20190367954A1 (en) | 2019-12-05 |
US10767198B2 US10767198B2 (en) | 2020-09-08 |
Family
ID=54849913
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/779,214 Active 2036-01-22 US10767198B2 (en) | 2015-11-28 | 2015-11-28 | Method for producing branched aldehydes |
Country Status (5)
Country | Link |
---|---|
US (1) | US10767198B2 (en) |
EP (1) | EP3380625B1 (en) |
JP (1) | JP6850292B2 (en) |
CN (1) | CN108474011A (en) |
WO (1) | WO2017088937A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021180327A1 (en) * | 2020-03-12 | 2021-09-16 | Symrise Ag | Methods for the biotechnological production of aldehyde mixtures |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4141124A1 (en) * | 2021-08-31 | 2023-03-01 | Symrise AG | Method for the biotechnological manufacture of aldehyde mixtures |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0286788A (en) | 1988-09-24 | 1990-03-27 | Nippon Oil & Fats Co Ltd | Production of fatty acid and hair tonic |
WO2007142784A1 (en) | 2006-05-31 | 2007-12-13 | Archer-Daniels-Midland Company | Enzymatic method of making aldehydes from fatty acids |
DE102010039833A1 (en) | 2010-08-26 | 2012-03-01 | Symrise Ag | Whole-cell biotransformation of fatty acids to the fatty aldehydes truncated by one carbon atom |
US9650653B2 (en) | 2011-06-30 | 2017-05-16 | Invista North America S.A.R.L. | Bioconversion process for producing nylon-7, nylon-7,7 and polyesters |
-
2015
- 2015-11-28 JP JP2018527761A patent/JP6850292B2/en active Active
- 2015-11-28 CN CN201580085674.9A patent/CN108474011A/en active Pending
- 2015-11-28 EP EP15808552.2A patent/EP3380625B1/en active Active
- 2015-11-28 WO PCT/EP2015/077992 patent/WO2017088937A1/en active Application Filing
- 2015-11-28 US US15/779,214 patent/US10767198B2/en active Active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021180327A1 (en) * | 2020-03-12 | 2021-09-16 | Symrise Ag | Methods for the biotechnological production of aldehyde mixtures |
Also Published As
Publication number | Publication date |
---|---|
JP2019500861A (en) | 2019-01-17 |
EP3380625B1 (en) | 2020-08-05 |
WO2017088937A1 (en) | 2017-06-01 |
JP6850292B2 (en) | 2021-03-31 |
US10767198B2 (en) | 2020-09-08 |
EP3380625A1 (en) | 2018-10-03 |
CN108474011A (en) | 2018-08-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Hanson | The chemistry of fungi | |
EP0258993B1 (en) | Production of lactones | |
Wińska et al. | Antimicrobial activity of new bicyclic lactones with three or four methyl groups obtained both synthetically and biosynthetically | |
US10767198B2 (en) | Method for producing branched aldehydes | |
US20070224668A1 (en) | Process for producing 4-vinylguaiacol by biodecaroxylation of ferulic acid | |
Kallel-Mhiri et al. | Mechanism of ethyl acetate synthesis by Kluyveromyces fragilis | |
US20180142270A1 (en) | Streptomyces psammoticus and methods of using the same for vanillin production | |
KR20010033413A (en) | Novel substances kf-1040 and process for producing the same | |
EP0356291B1 (en) | Process for the microbiological production of gamma-decalactone (R) and gamma-octalactone (R) | |
JP4114992B2 (en) | Method for producing tetrahydrocurcumin | |
EP0425001B1 (en) | Natural delta-lactones and process of the production thereof | |
US10196659B2 (en) | Method for producing lactones from a strain of Aureobasidium pullulans | |
ESmAEili et al. | Microbial transformation of citral by Aspergillus niger-PTCC 5011 and study of the pathways involved. | |
JP5174430B2 (en) | Method for producing 2-phenylethyl alcohol | |
US5620879A (en) | Process for producing natural cis-3-hexenol from unsaturated fatty acids | |
JPH06153924A (en) | Production of substituted methoxyphenol and microorganism suited for this purpose | |
JPH11221091A (en) | Production of fatty acid degradation product | |
JP2003144188A (en) | New method for producing xanthophyll educt and method for purifying the same | |
Seydametova | Selection of fermentation medium for fungal producer of pravastatin isolated from oil palm plantation soil | |
US6025170A (en) | Process for the biotechnological production of δ-decalactone and δ-dodecalactone | |
WO2004111204A1 (en) | Fungus strain and a method of obtaining mannitol from the same | |
JPH06116583A (en) | Fragrant substance and its production | |
US5219742A (en) | Method of producing gamma-hydroxydecanoic acid or its lactone by feeding a ricinoleic acid source to sp. odorus or rh. glutinis | |
CN107142220B (en) | Trichosporon for producing gamma-decalactone and application thereof | |
JPH0591889A (en) | Production of oil and fat and microorganism therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
AS | Assignment |
Owner name: SYMRISE AG, GERMANY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FRAATZ, MARCO A.;ZORN, HOLGER;ROST, JOHANNA;AND OTHERS;SIGNING DATES FROM 20180829 TO 20180917;REEL/FRAME:047192/0834 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: AWAITING TC RESP., ISSUE FEE NOT PAID |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 4 |